基于PTN系统分析不同种植地转基因水稻种子可培养内生真菌菌群的多样性

【目的】比较不同种植地、不同品种来源的转基因(genetically modified,GM)水稻近等基因系种子内生真菌的丰富度和多样性,探讨变异发生的影响因素,为研究GM水稻种子内生真菌种群结构的非预期变异提供科学基础。【方法】收集转2mG2-epsps抗草甘膦水稻株系(transgenic line,T),及相应的对照样本亲本品种(parent variety,P)和非转基因组培再生株系(non-transgenic regeneration line from tissue culture,NR),建立亲本对照-转基因株系-非转基因对照(parent control-transgenic plant line-non-transgenic control,PTN)近等基因系。粳稻品种日本晴亲本(P1)及其相应的T16和NR25组成P1近等基因系(P1 near-isogenic line,P1L),粳稻品种PJ574亲本(P2)及其相应的T23和NR18组成P2近等基因系(P2 near-isogenic line,P2L)。在海南省(Hainan province,H)和浙江省...

Diversity of Endophytic Fungi in Transgenic Rice Seeds from Different Planting Sites Based on PTN System

【Objective】To provide scientific bases for study of the unintended variation in seed endophytic fungi community structure of genetically modified (GM) rice and explore the causing factors of variation, comparative analysis of the richness and biodiversity of seed endophytic fungi were conducted, using GM rice relative near-isogenic lines of different varieties in different cultivation sites as materials. 【Method】 Collect transgenic rice line which harboring the glyphosate resistant 2mG2-epsps gene (T) and its corresponding parent variety (P) and non-transgenic tissue culture regeneration control line (NR) to make parent control-transgenic plant line-non-transgenic control (PTN) near-isogenic line. The parent japonica rice Nipponbare (P1) and its corresponding transgenic line T23 and the NR control line NR18 formed the P1 near-isogenic line (P1L), the P2 near-isogenic line (P2L) composed of parent japonica rice PJ574 (P2) and its corresponding transgenic line T23 and the NR control line NR18. All rice samples were planted in two plantations including Hainan province (H) and Fuyang city of Zhejiang province (F) and the resulting seeds were harvested. The rice endophytic fungi were isolated by tissue separation method, strains were classified and identified with morphology and molecular biology methods. The isolation rate (IR), isolation frequency (IF), richness Margalef index (D), diversity Shannon-Wiener index (H') and Evenness index (E) were used to reflect the structure and distribution of rice seed endophytic fungi, and Sorenson similarity coefficient (Cs) and Fisher’s exact test were employed to describe the composition difference of endophytic fungi between rice samples. 【Result】A total of 121 endophytic fungi strains were isolated from rice seed samples of P1L and P2L near-isogenic lines that harvested in Hainan (H) and Zhejiang (F) plantations, they were identified as 15 genera, of which Curvularia, Dendryphiella, Epicoccum, Fusarium were confirmed as the dominant flora, with Fusarium as the common dominant genus of both H and F plantations. The total RF (4.61%) of endophytic fungi from Zhejiang-grown rice seeds is 5.05 times than that of Hainan-grown rice samples (0.83%). The richness Margalef index (D=2.29), Shannon-Wiener diversity index (H'=1.63) and Evenness index (E=0.66) of endophytic fungi flora from rice seeds in F plantation were higher than that (D=1.67, H'=0.63, E=0.29) of samples in H plantation. The seed endophytic fungi communities of P1L and P2L in H plantation had a similarity coefficient of 0.615, and that was 0.737 in F plantation, showing moderate similarity, and the Fisher’s exact test analysis suggested that there were no significant difference (P>0.05) between them. The GM lines except HT16 showed moderate similarity to their corresponding parent controls referring seed endophytic fungi communities, with similarity coefficient ranged between 0.500 and 0.667. But compared with their corresponding parents, the GM rice lines showed notably unintended variations in IF and genus numbers of endophytic fungi, the variation direction and amplitude of IF varied between different plantations. Also, the GM line FT16 increased 2 additional fungi genera Phaeosphaeria and Nigrospora, HT23 and FT23 respectively added 1 genus Letendraea helminthicola and 2 genera including Curvularia and Cladosporium. These genera increasing variations of endophytic fungi in GM rice seeds were derived from transgenic insertion mutation, whose safety needs to be focused on. While the genera decreasing variations of endophytic fungi in GM rice seeds were derived from somaclonal variation of tissue culture, which were safer. The variation amplitude order of total IF of rice endophytic fungi were as follows. Difference between H and F plantations referring to P1 and P2 (30.58%)>Difference between P1 and P2 varieties (27.28%)>Variation of NR lines (23.14%)>Variation of GM lines (22.32%). The genus number variations of rice endophytic fungi ranked as follows. Difference between H and F plantations referring to P1 and P2 (9)>Difference between P1 and P2 varieties (8) = Variation of NR lines (8) = Variation of GM lines (8). 【Conclusion】Rice seeds have abundant and diverse endophytic fungi, with majority of the culturable stains belong to Ascomycetes. Composition of rice seed endophytic fungi community shows geographical differences, and Fusarium is very common dominant genus in rice seeds grown both in Hainan and Zhejiang. The structure of endophytic fungi flora in rice seeds are affected by transgenic manipulation as well as tissue culture technology, while their unintended variation effects are less than that of rice growing locations and variety differences. The genera increasing variations of endophytic fungi in GM rice seeds are derived from transgenic insertion mutation, and the safety needs to be assessed.